US5770720A - Ubiquitin conjugating enzymes having transcriptional repressor activity - Google Patents
Ubiquitin conjugating enzymes having transcriptional repressor activity Download PDFInfo
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Definitions
- the present invention relates to a novel mammalian ubiquitin conjugating enzyme, and more particularly, to the identification, isolation, and purification of a human ubiquitin conjugating enzyme, the complete amino acid sequence of which has been elucidated, and to nucleotide sequences encoding the enzyme.
- the invention further relates to novel methods of using the enzyme and similar enzymes to regulate gene transcription and, particularly to suppress transcription of a target gene in a human and non-human host cells.
- the invention relates to methods for enhancing the repressor activity of WT1, Wilm's tumor suppressor gene product.
- Ubiquitin has been identified as playing a central role in tagging proteins for degradation, and thus in modulating their life-span in the cell.
- nuclear proteins that are known to be regulated by ubiquitination include NFxb, cyclin B, c-jun, p53 and histones.
- Ubiquitin conjugating enzymes (UBCs) activate and attach ubiquitin to a protein targeted for degradation in the proteolytic proteosome pathway by transferring activated ubiquitin in thioester linkage. At least twelve separate yeast ubiquitin conjugating enzymes have been identified and sequenced.
- Tumor suppressor genes such as the p53 gene, the retinoblastoma (Rb) gene and the Wilm's tumor suppressor gene, encode proteins which inhibit cell reproduction and/or transcription in various ways.
- p53 gene protein is believed to bind to DNA and induce transcription of another regulatory gene, the product of which blocks the kinase activity of proteins important for normal cell cycle progression, thereby precluding cell replication.
- the Rb gene protein is thought to act by masking the activation domain of an activator protein.
- a Rb gene product protein and a method therapeutic use thereof are disclosed in U.S. Pat. No. 5,496,731 to Benedict et al.
- Gene suppressor proteins may also act in other ways, including, for example, by competing with activator proteins for specific DNA binding sites, and/or by direct or indirect interaction with the general transcription factors.
- Other tumor suppressor genes and gene products are disclosed in U.S. Pat. No. 5,491,064 to Howley et al. (HTS-1 gene).
- WT1 Wilm's Tumor (WT) suppressor gene product
- WT1 is a bifunctional transcription factor of the Kruppel zinc-finger family. Loss of function of both alleles of the WT1 gene (11p13) is associated with some Wilm's tumors and associated syndromes.
- WT1 is a 52 ⁇ 57 kd nuclear protein which contains a glutamine/proline-rich N-terminal region and four zinc-fingers of the C2-H2 subclass in the C-terminal region.
- WT1 is a potent repressor of the promoter activity of several growth related genes, including the IGF-II, PDGF A-chain, CSF-1 and IGF-R promoters.
- WT1 has an independent repressor domain which is active when WT1 interacts with DNA through the zinc-finger domains. While the activity of repressor gene products such as WT1 is known to affect transcription, control over the biochemical mechanism by which transcriptional repression is effected is not thoroughly understood and higher levels of repression are desirable for commercial applications.
- the present invention is directed to a novel, isolated and substantially purified mammalian ubiquitin conjugating enzyme, hUBC-9, having a molecular weight of about 16 kilodaltons to about 18 kilodaltons, preferably about 17 kilodaltons, a sequence length of from about 150 to 165 amino acid residues, preferably 158 amino acid residues, and having conjugating activity and/or transcriptional repressor activity.
- the present invention is also directed to a protein having an amino acid sequence which includes the amino acid sequence of hUBC-9, SEQ ID NO: 3.
- the invention is directed as well to a protein which has ubiquitin conjugating activity or transcriptional repressor activity and which includes a portion of the amino acid sequence of hUBC-9, SEQ ID NO: 3, at least about 12 amino acid residues in length.
- the included portion of the hUBC-9 sequence confers the conjugating activity or the repressor activity on the protein.
- the invention is directed to proteins which have transcriptional repressor activity and have at least about 60% sequence identity to hUBC-9, SEQ ID NO: 3, more preferably at least about 65% sequence identity, more preferably at least about 75% sequence identity, more preferably at least about 85% sequence identity and most preferably at least about 95% sequence identity to hUBC-9.
- a C 93 mutant of hUBC-9, which does not have ubiquitin conjugating activity, but retains its transcriptional repressor activity, is a particularly preferred protein.
- the invention is directed, moreover, to substantially isolated nucleic acid polymers encoding hUBC-9.
- the nucleic acid polymer preferably has a nucleic acid sequence selected from the group consisting of: (a) SEQ ID NO: 1; (b) SEQ ID NO: 2; (c) a nucleic acid sequence which includes the nucleic acid residues defined by the sequence from position 88 to position 564 of SEQ ID NO: 2.
- the invention is also directed to a substantially isolated nucleic acid polymer which encodes a protein having ubiquitin conjugating activity or transcriptional repressor activity and having an amino acid sequence which includes a portion of the amino acid sequence of hUBC-9, SEQ ID NO: 3.
- the included portion is at least about a 12 amino acid residues in length and confers the conjugating activity or the repressor activity on the protein.
- the invention is directed to a nucleic acid polymer which is at least about 36 nucleic acid residues in length and which encodes a protein which has transcriptional repressor activity.
- Such a nucleic acid fragment can encode a protein which has at least about 60% sequence identity to hUBC-9SEQ ID NO: 3, or alternatively, can hybridize to a nucleic acid polymer which is complementary to the aforementioned nucleic acid polymers which constitute a part of the invention.
- the invention is also directed to nucleic acid polymers which are complementary to the aforementioned nucleic acid polymers of the invention.
- the invention is directed as well to methods for producing hUBC-9 or a segment thereof using a host cell transfected with a vector having a DNA which encodes hUBC-9 or a segment thereof.
- the method preferably comprises producing a plasmid vector having DNA (including genomic DNA and/or genomic DNA).
- the DNA encodes the aforementioned hUBC-9 protein or a segment or homolog thereof.
- the plasmid vector is transfected into a host cell and hUBC-9 is expressed in the host cell. If desired, the expressed hUBC-9 may be purified from the host cell.
- the invention is also directed to the vector and to the host cell transfected therewith.
- the invention is further directed to a host cell co-transfected with first and second plasmid vectors each comprising DNA.
- the DNA of the first vector comprises a nucleic acid polymer which encodes a transcriptional repressor protein other than a UBC-9 protein, including for example, WT1.
- the DNA of the second vector comprises a nucleic acid polymer which encodes an adapter protein having transcriptional repressor activity which is preferably independent of the transcriptional repressor activity of the transcriptional repressor protein.
- the adapter protein associates or interacts with the transcriptional repressor protein after both are co-expressed in the host cell.
- the adapter protein has an amino acid sequence which includes a portion of the amino acid sequence of a ubiquitin conjugating enzyme that has transcriptional repressor activity.
- ubiquitin conjugating enzymes include hUBC-9, yUBC-9, other members of the UBC-9 family and other ubiquitin conjugating enzymes.
- the included portion of the amino acid sequence is at least about 12 amino acid residues in length.
- the invention is directed, moreover, to a fusion protein comprising a transcriptional repressor domain and a DNA binding domain.
- the transcriptional repressor domain has an amino acid sequence which includes at least a 12 amino acid residue portion of the amino acid sequence of a ubiquitin conjugating enzyme that has transcriptional repressor activity.
- the DNA binding domain is preferably a domain which binds sufficiently close to a promoter region of a target gene to allow the ubiquitin conjugating enzyme to repress transcription.
- Exemplary DNA binding domains include Gal4, LexA and any of the zinc-finger domains.
- the invention also relates to nucleic acid polymers encoding such a fusion protein, plasmid vectors comprising such nucleic acid polymers and to host cells transformed therewith.
- the invention is directed as well to a method for producing a fusion protein having a transcriptional repressor domain and a DNA-binding domain.
- the method comprises: producing a plasmid vector comprising DNA which encodes the fusion protein described above, transfecting the plasmid vector into a host cell, expressing the fusion protein in the host cell, and, if desired, purifying the expressed fusion protein from the host cell.
- the invention is directed to a composition
- a composition comprising a protein having transcriptional repressor activity and an acceptable carrier, diluent or biochemical delivery agent suitable for introducing the protein into a target cell.
- the protein has transcriptional repressor activity and has an amino acid sequence which includes at least a 12 amino-acid residue long portion of a ubiquitin conjugating enzyme which has transcriptional repressor activity.
- the protein derives its transcriptional repressor activity from the included portion of the enzyme.
- the composition can be used for non-pharmaceutical (ie, non-human) uses, but can also be a pharmaceutical composition, in which the aforementioned protein is combined with a pharmaceutically acceptable carrier, diluent and/or gene therapy delivery agent.
- the invention is further directed to a composition suitable for use in introducing a nucleic acid polymer to a cell, whereby the expression product of the nucleic acid polymer is exposed to and/or contacts a target gene therein.
- the composition can be used for pharmaceutical or non-pharmaceutical applications to regulate transcription.
- the composition comprises a nucleic acid polymer and a gene therapy delivery agent.
- the amount of the nucleic acid polymer or construct containing the same is sufficient to, upon expression in a host cell, express a pharmaceutically effective amount of the protein to regulate a target gene in the cell.
- the nucleic acid polymer is used in conjunction with a pharmaceutically acceptable gene therapy delivery agent.
- the nucleic acid polymer encodes a protein having transcriptional repressor activity and having an amino acid sequence which includes at least a 12 amino-acid residue long portion of a ubiquitin conjugating enzyme which has transcriptional repressor activity. The included portion of the enzyme confers the repressor activity on the protein.
- the nucleic acid polymer can, alternatively, have a nucleic acid sequence which is complementary to the aforementioned nucleic acid polymers of the composition.
- the nucleic acid composition can be a virus which has a viral genome that includes the nucleic acid polymer being delivered to the target gene.
- the ubiquitin conjugating enzyme used in such a composition is preferably a UBC-9 protein such as hUBC-9, yUBC-9 or yUBC-9-m.
- Another pharmaceutical composition comprises a pharmaceutically active amount of the fusion protein set out above and a pharmaceutically acceptable carrier.
- the invention is also directed to a method of regulating transcription of a target gene in a cell.
- the method comprises exposing the target gene to and/or contacting the target gene with a protein having transcriptional repressor activity.
- the protein has an amino acid sequence which includes at least a 12 amino acid residue portion of the amino acid sequence of a ubiquitin conjugating enzyme having transcriptional repressor activity, such as a UBC-9.
- a composition which includes the protein or which includes a nucleic acid polymer encoding the protein may be introduced into the cells in a number of ways, including by contacting, infecting or transfecting the target cells with a gene therapy delivery agent such as a virus.
- the amount of protein to which the gene is exposed is more than the endogenous amount normally present within the cell.
- the cell can be an eukaryotic cell such as a fungal cell (e.g. a yeast cell), a plant cell, a non-human animal or mammalian cell or a human cell.
- the cell can also be a cell which has been infected with a virus wherein the viral genome is exposed to the transcription regulating protein.
- the invention is also directed to method of modulating neoplastic tissue growth. In the method, neoplastic tissue cells are contacted with a neoplastic-tissue-growth-modulating amount of one of the pharmaceutical compositions set forth above, thereby modulating the growth of the neoplastic tissue.
- the invention relates as well to a method of inhibiting the proliferation of Wilm's tumor cells.
- the method comprises introducing into Wilm's tumor cells a Wilm's-tumor-inhibiting amount of a ubiquitin conjugating enzyme or a segment thereof having transcriptional repressor activity, or alternatively, introducing a nucleic acid polymer which encodes such an amount of the enzyme, and preferably co-expressing WT1 therewith.
- hUBC-9 and other members or the UBC-9 family can be used, for example, in chemotherapy, gene therapy and drug development.
- Other uses of the invention include its use to regulate the rate of both specific and general gene transcription and the cell cycle, including the control of abnormal expression of genes associated with human disease such as those caused by virus, or associated with yeast infections.
- the invention has use in non-pharmaceutical applications in the yeast, baking and brewing industries, and also in conjunction with enzymatic conversion methods for producing valuable chemical commodities such as essential amino acids.
- the enzyme, its activities and other features, methods of expressing the enzyme, and methods for its use are described in greater detail below.
- FIGS. 1A and 1B show nucleotide and amino acid sequences for hUBC-9.
- FIG. 1A shows the full length nucleotide sequences (SEQ ID NO: 1 and SEQ ID NO: 2) and the predicted amino acid sequence (SEQ ID NO: 3) for two cDNA clones encoding hUBC-9.
- the vertical line connecting the cytosine at position 792 of the longer form and at position 73 of the shorter form indicates the splice site and origin of common nucleotide sequences of the two alternative spliced mRNA.
- FIG. 1B shows a comparison of predicted amino acid sequences of hUBC-9 (SEQ ID NO: 3) and yUBC-9(SEQ ID NO: 4).
- FIGS. 2A and 2B show the results of Northern and Southern blot analyses, respectively.
- FIG. 2A shows Northern blot of hUBC-9 in different human tissues.
- FIG. 2B shows Southern blot analysis of the hUBC-9 gene.
- FIGS. 3A and 3B show in vitro binding of WT1 and hUBC-9, in Western blot analyses of associated WT1 and hUBC-9 proteins.
- FIG. 3A shows blots of eluates taken from matrix-coupled GST-hUBC-9 which had been passed over and incubated with WT1 from 293 cell extracts.
- FIG. 3B shows the results of co-immunoprecipitation of WT1 and hUBC-9 from 293 cells co-transfected with WT1 and HA-tagged hUBC-9 expression vectors.
- FIGS. 4A and 4B show temperature-sensitive yeast cell cultures at permissive and restrictive temperatures, respectively.
- FIGS. 5A and 5B show how hUBC-9 enhances the transcriptional repressor activity of WT1 in human embryonic kidney cells (293).
- FIG. 5A shows the relative CAT activity when various amounts of WT1 and hUBC-9 expression vectors are co-transfected.
- FIG. 5B illustrates the relative CAT activity of independent assays at different times ⁇ standard deviation of each.
- FIGS. 6A and 6B show the transcriptional repressor activity of a hUBC-9/Gal4 DNA binding domain fusion protein.
- FIG. 6A shows the expression and reporter vector constructs.
- FIG. 6B shows the relative CAT activity.
- FIGS. 7A-7C show the transcriptional repressor activity of hUBC-9, yUBC-9, and yUBC-9-m/Gal4 DNA binding domain fusion proteins.
- FIG. 7A shows the expression and reporter vector constructs.
- FIG. 7B shows the relative CAT activity for hUBC-9/Gal4 fusion proteins.
- FIG. 7C shows the relative CAT activity for yUBC-9/Gal4 and yUBC-9-m/Gal4 fusion proteins.
- FIG. 8 shows the results of GST/hUBC-9 capture assays for TATA binding protein (TBP), transcription factor IIB (TFIIB) and Wilm's tumor suppressor gene product, WT1.
- FIGS. 9A and 9B relate to GST/hUBC-9 capture assays with wild type hTBP and with several mutant TATA binding proteins, mTBP.
- the shaded area of the wild-type hTBP represents the highly conserved region between species.
- the shaded area for the mutant TBP's represents the portion of the TBP deleted.
- the term "dl x-y" indicates that in the mTBP, residues x through y were deleted.
- FIG. 9B shows the blots resulting from the various GST/hUBC-9 capture assays.
- FIGS. 10A-10D show the results of WT1 turnover experiments done when WT1 is expressed by itself (FIG. 10A), when WT1 is co-expressed with hUBC-9 (FIG. 10B), when WT1 is expressed in the presence of lactocysteine, a known inhibitor of the proteolytic degradation system (FIG. 10C) and when WT1 is co-expressed with mUBC-9, a C 93 S mutant of hUBC-9 (FIG. 10D).
- FIGS. 11A and 11B relate to gel mobility shift assays that show the interaction between hUBC-9 and the TATA binding protein (TBP).
- FIG. 11A shows the results of assays using hUBC-9 and an end-labeled DNA probe containing the TATA box either (a) without TBP present (columns A1-A3), (b) with TBP present but without TFIIB present (columns B1-B3) and (c) with both TBP and TFIIB present (columns C1-C3).
- FIG. 11B shows the results of similar assays in which TBP was present with varying amounts of hUBC-9.
- FIGS. 12A and 12B show the results of transient co-transfection assays using a 5 ⁇ UAS pSV CAT reporter vector.
- FIG. 12A shows the relative level of expression of the reporter vector for assays where a GAL4/hUBC-9 fusion protein (pSGhUBC-9) (0 or 10 ⁇ g), TBP (0, 0.5 or 2.5 ⁇ g) and/or TFIIB (5 ⁇ g) were co-expressed in 293 cells in varying combinations.
- FIG. 12B shows the relative level of expression of the reporter vector where a mutant TBP, TBPA1-138 (0, 2.5 and 5 ⁇ g), and the hUBC-9/Gal4 fusion protein (0, 10 ⁇ g) were co-expressed in various combinations.
- FIG. 13 shows the results of transient co-transfection assays using a 5 ⁇ UAS pSV CAT reporter vector where TBP ⁇ 1-138 (0, 2.5 and 5 ⁇ g) was co-expressed with WT1 (10 ⁇ g) in various combinations.
- a “substantially purified” protein means that the protein is separated from a majority of host cell proteins normally associated with it or that the protein is synthesized in substantially purified form, such synthesis including expression of the protein in a host cell from a nucleic acid polymer exogenously introduced into the cell by any suitable gene-therapy delivery means.
- a “substantially isolated” nucleic acid polymer means that the mixture which comprises the nucleic acid polymer of interest is essentially free of a majority of other nucleic acid polymers normally associated with it.
- a “nucleic acid polymer” includes a polymer of nucleotides or nucleotide derivatives or analogs, including for example deoxyribonucleotides, ribonucleotides, etc.
- Genomic DNA, cDNA and mRNA are exemplary nucleic acid polymers.
- the term “regulate transcription” is intended to include enhancement and/or repression of transcription.
- the term “gene” is intended to include both endogenous and heterologous genes, and specifically, both genomic DNA which encodes a target protein in a naturally occurring cell, and also cDNA encoding the target protein, wherein the CDNA is a part of a nucleic acid construct such as a plasmid vector or virus which has been introduced into a cell.
- the present invention relates to newly discovered human ubiquitin conjugating enzymes, designated hUBC-9, which in addition to having a functional conjugating activity, have an independent transcriptional repressor activity. Both the conjugating and the repressor activities have been found to influence transcription.
- the conjugating activity of hUBC-9 enhances transcription through degradation of transcription suppressor proteins such as WT1, and possibly, of hUBC-9 itself.
- the repressor activity of hUBC-9 represses transcription independently of the conjugating activity.
- hUBC-9 strikingly enhances the function of WT1 as a repressor of gene transcription.
- the enzyme also acts independently of WT1 to suppress gene transcription itself, particularly when fused to proteins having a DNA binding domain, such as Gal4.
- UBC-9 acts as a potent repressor by disrupting the transcriptional initiation complex through specific interactions with the DNA binding region of the TATA binding protein (TBP). Such interactions are concentration dependent and result in destabilized TPB/DNA interactions and interference with formation of the TFIIB/TBP transcription initiation complex.
- hUBC-9 can operate in conjunction with other proteins having a repressor effect, such as WT1, to result in a combined repressor effect which is enhanced relative to the repressor effect of WT1 alone or of hUBC-9 alone.
- hUBC-9 association of hUBC-9 with other repressor proteins, either through protein-protein interactions as with WT1 or by positioning hUBC-9 in the vicinity of the promoter as a fusion protein comprising hUBC-9 and a DNA binding domain, such as the domain of Gal4, Lex A, zinc-fingers or others.
- DNA binding proteins which are fused to hUBC-9 or repressor proteins which specifically interact with hUBC-9 appear to position human UBC-9 to an appropriate site in relation to promoter DNA such that hUBC-9 can interact with the TBP, and thereby reduce transcription initiation.
- the conjugating activity of hUBC-9 appears to operate in conjunction with hUBC-9's repressor activity by regulating the levels of WT1 present in the system.
- Such regulation of WT1 levels is accomplished through its ubiquitin conjugating activity and the associated ubiquitin-dependent proteolytic pathway.
- the ubiquitin conjugating activity of hUBC-9 and its repressor activity may act at the same time, with hUBC-9 interacting simultaneously with WT1 (via its conjugating activity) and with the TBP (via its repressor activity).
- hUBC-9 may also interact with other repressors such as p53 and Rb in a similar manner.
- inhibition of UBC-9's conjugating activity results in even a greater degree of repression of the transcription initiative.
- the homologous ubiquitin conjugating enzymes of other eukaryotes exhibit the same bifunctional activities as hUBC-9: transcription repression and ubiquitin conjugation.
- hUBC-9 transcription repression and ubiquitin conjugation.
- UBC-9 includes any such proteins whether they are identified herein or discovered in the future.
- hUBC-9 for humans and yUBC-9 for yeast.
- Many facets of the invention also relate to other ubiquitin conjugating enzymes other than the UBC-9 enzymes, provided, that such ubiquitin conjugating enzymes have transcriptional repressor activity in addition to their conjugating activity.
- the several aspects of the present invention including the hUBC-9 protein and nucleic acid polymers which encode it, the transcriptional repressor activity of ubiquitin conjugating enzymes such as a UBC-9 enzyme, and the interaction between UBC-9 enzymes and other transcription repressor proteins, especially such proteins having a DNA binding domain, and in particular tumor suppressor proteins such as WT1, collectively enable several practical applications, including both pharmaceutical applications involving humans and non-pharmaceutical uses.
- FIG. 1A shows the complete nucleotide sequences of two independent cDNA clones, designated as SEQ ID NO: 1 and SEQ ID NO: 2, which were established from two alternatively spliced mRNAs.
- the cDNA clones both encode hUBC-9.
- the amino acid product resulting from transcription and translation of these cDNA clones migrated identically in SDS-containing polyacrylamide gel as a 17 kilodalton protein.
- hUBC-9 has the amino acid sequence set forth in SEQ ID NO: 3 and shown in FIG. 1B. Based on a comparison with data in Genebank, hUBC-9 is an active human (h) homolog of the yeast ubiquitin conjugating enzyme-9, yUBC-9 or E2, the intermediate enzyme in the ubiquitin protein degradation pathway.
- the human UBC-9 sequence has a 56% amino acid identity overall with yUBC-9, including identical sequences of 9 amino acids in separate regions.
- the 158 amino acid sequence of hUBC-9 also contains a cysteine residue in precise alignment with the active site cysteine of yeast UBC-9 (boxed, FIG. 1B).
- Human UBC was expressed in a all human tissues tested, including heart, brain, placenta, lung, smooth muscle, kidney and pancreas tissues. (Example 2). However, the level of expression varied in different tissues, as demonstrated by the results of a Northern blot experiment shown in FIG. 2A. Northern blots of hUBC-9 in different tissues resulted in generally strong hybridization signals of 2.8 and 1.3 kb. However, heart and smooth muscle are seen to express significantly higher levels of transcripts relative to other tissues analyzed, and kidneys appear to express relatively less of the 2.8 kb mRNA isoform.
- hUBC-9 is encoded by a single gene, as demonstrated by experiments in which Southern blots of human genomic DNA were digested with different restriction enzymes and probed with the 1.1 kb fragment of hUBC-9 cDNA. (Example 3). Single hybridization signals were seen in digests of PstI and BamHI (FIG. 2B), suggesting that the human UBC gene exists as a single copy gene in the human genome.
- hUBC-9 is further characterized by its association with the repressor domain of the Wilm's tumor suppressor gene product, WT1.
- Human UBC binds to WT1 both in vivo and in vitro. Protein-protein interactions between hUBC-9 and WT1 were initially demonstrated in the yeast two hybrid system by high levels of ⁇ -galactosidase activity, as shown in Table 1. (Example 1). Additional experiments were carried out to confirm the WT1-human UBC protein interactions observed in yeast. Human UBC was expressed as a glutathione S-transferase fusion protein (GST-human UBC) in E.coli and coupled to a glutathione matrix. (Example 4). As shown in FIG.
- hUBC-9 also interacts directly with the TATA-binding protein (TBP), as demonstrated by GST capture assays.
- TBP TATA-binding protein
- a GST fusion protein having the full-length hUBC-9 amino acid sequence captured TBP and WT1 selectively over the transcriptional factor TFIIB, which was also present in the assay.
- FIG. 8 Further assays demonstrated that hUBC-9 interacts with the TBP through the highly conserved C-terminal domain of the TBP.
- Several mutants of hTBP were constructed. (FIG. 9A).
- GST/hUBC-9 captured wild-type TBP as well as several of the mutant TPB's; however, deletion of the C-terminal region of TBP (amino acid residues 196-335) significantly reduced the capture efficiency, and deletion of a larger portion thereof (amino acid residues 163-335) resulted in no interactions being detectable in the assay. (FIG. 9B).
- hUBC-9 specifically interacts with the C-terminal domain which includes amino acids 163-335 of the TATA binding protein.
- Gel mobility shift assays discussed below, further confirmed the specificity of the interaction between TBP and hUBC-9.
- the hUBC-9 Enzyme has an Active Conjugating Activity
- hUBC-9 is an active ubiquitin conjugating enzyme, and as such, is a member of a family of ubiquitinating enzymes.
- the active cysteine residue at position 93 (boxed, FIG. 1B) is characteristic of all of the ubiquitin conjugating enzymes discovered to date, and it has been determined that the presence of the active site cysteine is important to the ubiquitin conjugating activity.
- This cysteine is believed to provide the enzyme with its ability to participate in thioester formation.
- human UBC-9 which has 56% sequence identity to yeast UBC-9 and shares the same active cysteine site, forms an integral part of the proteolytic proteosome pathway.
- the human ubiquitin conjugating protein of the present invention is a member of a family of enzymes which, via their conjugating activity, function to regulate the cell cycle and duplication of DNA. It has now been determined, moreover, that the ubiquitin-dependent protease degradation system is directly involved in transcriptional regulation.
- the conjugating activity of hUBC-9 appears to modulate gene transcription by contributing to the degradation of repressor proteins such as WT1, thereby regulating the level of repressor activity.
- WT1 was shown to be rapidly degraded by the ubiquitin proteosome proteolysis pathway when expressed in rabbit reticulocyte lysates containing the enzymes required for transiting the proteolysis pathway.
- cDNA of WT1 was added to rabbit reticulocyte lysates that contained E1, E2 and E3 enzymes, ubiquitin and the 26S proteosome complex required for protein ubiquitination and degradation.
- a distinct band that migrated identically to WT1 was observed upon analysis in SDS gels.
- cDNAs of both WT1 and hUBC-9 were added to the rabbit reticulocyte system, one protein band had a greatly diminished intensity relative to the band observed in the control experiment.
- the effect of the conjugating activity is also demonstrated by inhibition of the proteolytic pathway with lactocysteine, a specific inhibitor of protease activity associated with the 26S proteosome complex, with such inhibition causing the half-life of WT1 to increase.
- the turnover of WT1 was tested in a series of experiments in which WT1 was (a) expressed in 293 cells alone, (b) co-expressed with hUBC-9, (c) expressed in the presence of lactocysteine, a known inhibitor of the proteolytic degradation system or (d) co-expressed with mUBC-9, a C 93 S mutant of hUBC-9. In each case, the cells were treated with cycloheximide, a protein synthesis inhibitor.
- WT1-dependent transcriptional repression is influenced by the proteosome degradation system.
- WT1 was expressed in 293 cells co-transfected with a reporter plasmid and cultured with lactocysteine (50 ⁇ M)
- lactocysteine 50 ⁇ M
- a two-fold to three-fold enhancement of repressor activity was observed.
- the enhancement effect of lactocysteine progressively decreased as the level of expression of WT1 was increased. Lactocysteine was without effect at an upper limiting level of WT1.
- the conjugating activity of hUBC-9 and its effect on repression is independently demonstrated by removal of the conjugating activity.
- hUBC-9 has conjugating activity which is specific to the WT1 and possibly to other suppressor proteins.
- the conjugating activity of hUBC-9 positively influences transcription through degradation of repressors such as WT1, and possibly, of hUBC-9 itself.
- Ubiquitin conjugating enzymes such as hUBC-9 and yUBC-9 have a transcriptional repression activity.
- the repression activity of hUBC-9 enhances the existing repressor activity of WT1 and perhaps other repressor gene products.
- hUBC-9 enzyme strikingly enhances the repressor activity of Wilm's tumor suppressor gene product, WT1.
- the ability of human UBC to modulate the transcriptional regulatory activity of WT1 was analyzed in cotransfection experiments (Example 7). As shown in FIG. 5A, when human UBC was expressed alone at 15 ⁇ g, without WT1 and without being fused to a DNA binding domain, expression of the reporter gene was reduced slightly more than 2-fold (42% relative activity). When WT1 was expressed alone at 10 ⁇ g, expression of the reporter gene was reduced by about a factor of ten (8% relative activity).
- hUBC-9 is itself a potent transcriptional repressor when it is coupled to a functional DNA binding domain recognized by an appropriate promoter element.
- Human UBC was coupled to the Gal4 DNA binding domain and tested with a promoter containing five upstream Gal4 DNA binding sequences (5 ⁇ UAS) in co-transfection experiments. (Example 8). As shown in FIG. 6B, when human UBC was tested with the control promoter (lacking 5 ⁇ UAS) reporter plasmid to which human UBC was unable to bind, the influence of human UBC was minimal (66% relative activity).
- human UBC was a powerful transcriptional repressor when it was able to directly bind, via the Gal4 binding domain, to the promoter/reporter construct containing the Gal4 DNA binding sequences.
- human UBC repressed promoter activity by about eight-fold (12% relative activity), establishing that human UBC alone is an effective repressor when it binds to an appropriate promoter element.
- Example 9 the h-UBC-9/Gal4 fusion protein again demonstrated significant repression activity (15% relative activity), as shown in FIG. 7B.
- hUBC has transcriptional repressor activity.
- the repressor activity of hUBC is particularly significant when hUBC is positioned near the promoter regions, either through protein-protein interactions with other proteins, such as WT1, or through fusion with DNA binding domains, such as Gal4, both of which appear to tether human UBC to gene-specific promoter sites.
- ubiquitin conjugating enzymes such as yUBC-9
- yUBC-9 also function efficiently as transcription repressors when fused to a DNA binding domain.
- Yeast UBC-9 was coupled to the Gal4 DNA binding domain and co-expressed with a reporter vector having five upstream Gal4 DNA binding sequences. (Example 9). As shown in FIG. 7C, when co-expressed by itself at 20 ⁇ g, the yUBC-9/Gal4 fusion protein repressed transcription by about three-fold (0.35 relative activity).
- UBC's have, in addition to their ubiquitin conjugating activity, a transcriptional repression activity.
- the repression activity is independent of the conjugating activity, as demonstrated by data showing that yUBC-9-m, a yUBC-9 C 93 S mutant which lacks ubiquitin conjugating activity, functions efficiently as transcription repressor when fused to a DNA binding domain.
- yUBC-9-m a mutant form of yUBC-9 lacking the active site cysteine at position 93 (designated yUBC-9-m) was coupled to the Gal4 DNA binding domain and co-expressed with a reporter vector having five upstream Gal4 DNA binding sequences. (Example 9).
- the yUBC-9-m/Gal4 fusion protein repressed transcription by about five-fold (20% relative activity).
- the ubiquitin conjugating activity does not appear to be required for the transcription repressor activity. Nonetheless, as discussed above, the conjugating activity of UBC-9 appears to affect and regulate the level of repressor activity.
- the conjugating activity of hUBC-9 facilitates proteolytic degradation of WT1 and thereby at least partially relieves the repressor effect of WT1.
- hUBC-9 Functions as a Repressor Through its Interactions with the TATA Binding Protein (TBP)
- hUBC-9 interacts with the C-terminal domain of the TATA binding protein (TBP) in GST capture assays.
- TBP TATA binding protein
- Gel mobility shift assays confirmed this interaction, and further demonstrated that hUBC-9 appears to suppress transcription by disrupting the binding of TBP to DNA and by disrupting the formation of the transcription initiation complex.
- This model is consistent with the understanding that the C-terminal of TBP, with which hUBC-9 was shown to interact, contains a "face" which contacts the major groove of DNA.
- an end-labeled DNA probe containing the TATA box was provided an opportunity to complex with various combinations of TBP, TFIIB and hUBC-9.
- hUBC-9 reduced the level of complex formation between TBP and DNA (FIG. 11A, columns B2 and B3), and in subsequent experiments, between TBP and DNA in the presence of TFIIB (FIG. 11A, columns C2 and C3).
- hUBC-9 destabilizes TBP/DNA binding.
- the effect of hUBC-9 on the DNA binding ability of TBP was concentration dependent. (FIG. 11B).
- hUBC-9 interacts in the region of the TBP DNA binding domain
- co-transfection assays were performed which demonstrated that high levels of exogenous TBP overcame the repressor activity of hUBC-9.
- a 5 ⁇ UAS pSV CAT reporter vector was used in transient assays in which a GAL4/hUBC-9 fusion protein (pSGhUBC-9) (0 or 10 ⁇ g), TBP (0, 0.5 or 2.5 ⁇ g) and/or TFIIB (5 ⁇ g) were co-expressed in 293 cells in varying combinations.
- the repressor activity of hUBC-9 was significantly reduced in a concentration-dependent manner by the presence of TBP (without TFIIB).
- TFIIB TFIIB
- FIG. 12A columns A, B, C and D
- TFIIB TFIIB
- FIG. 12A columns E and F
- Further experiments were performed to ensure that the observed decrease in repressor activity of hUBC-9 was not, in fact, related to an intrinsic activation of transcription due to expression of TBP.
- a mutant TBP was constructed which had an amino acid sequence which included the TBP domain which interacted with hUBC-9 in GST capture assays, but which did not include the amino acid residues 1-138.
- TBP ⁇ 1-138 The mutant TPB (0, 2,5 and 5 ⁇ g), referred to herein as TBP ⁇ 1-138, and the hUBC-9/Gal4 fusion protein (0, 10 ⁇ g) were co-expressed in various combinations in transient co-transfection assays similar to those immediately aforementioned.
- TBPA1-138 lacked the ability to substantially activate the promoter, but effectively relieved the repressor activity of hUBC-9, although to a lesser extent than wild-type TBP. (FIG. 12B).
- hUBC-9 is shown to interact with TBP to repress transcription.
- the addition of TBP can effectively titrate hUBC-9 to relieve its repressor activity, and in effect, enhance transcription.
- mutant TBP, TBP ⁇ 1-138 effectively relieved the repressor activity of WT1.
- FIG. 13 Cumulatively, these results suggest that WT1 effects suppression in combination with hUBC-9 by positioning hUBC-9 through protein-protein interactions for direct hUBC-9 interaction with the TBP subunit of the TFIID transcription factor, thereby destabilizing the TBP/TATA sequence interaction, and more generally, disrupting formation of the transcription initiation complex.
- the nucleotide sequence encoding the mammalian or yeast or other UBC enzyme, or active portion thereof, is cloned into an expression vector using known procedures. Briefly, specific nucleotide sequences in the vector are cleaved by site-specific restriction enzymes such as NcoI and HindIII. Then, after optional alkaline phosphatase treatment of the vector, the vector and a target fragment comprising the nucleotide sequence of interest are ligated together with the resulting insertion of the target codons in place adjacent to desired control and expression sequences.
- site-specific restriction enzymes such as NcoI and HindIII
- a host-compatible plasmid will be used containing genes for markers such as ampicillin or tetracycline resistance, and also containing suitable promoter and terminator sequences.
- a preferred plasmid into which the recombinant DNA expression sequence of the present invention may been ligated is plasmid pET.
- a pET plasmid which expresses human UBC-9 has been deposited in GeneBank, Ascession No.'s ⁇ 66818 and ⁇ 66867.
- the plasmid comprising the DNA expression sequence for the UBC enzymes of the present invention may then be expressed in a host cell.
- Bacteria e.g., various strains of E. coli, and yeast, e.g., Baker's yeast, are most frequently used as host cells for expression of mammalian UBC enzymes, although techniques for using more complex cells are known. See, e.g., procedures for using plant cells described by Depicker, A., et al., 1982.
- E. coli host strain X7029, wild-type F - , having deletion X74 covering the lac operon is utilized in a preferred embodiment of the present invention.
- a host cell is transformed using a protocol designed specifically for the particular host cell. For E.
- E. coli a calcium treatment produces the transformation. (Cohen, S. N., 1972). Alternatively and more efficiently, electroporation of salt-free E. coli is performed according to the method of Dower et al., 1988. After transformation, the transformed hosts are selected from other bacteria based on characteristics acquired from the expression vector, such as ampicillin resistance, and then the transformed colonies of bacteria are further screened for the ability to give rise to high levels of isopropylthiogalactoside (IPTG)-induced thermostable DNA polymerase activity. Colonies of transformed E. coli are then grown in large quantity and expression of mammalian UBC enzyme is induced for isolation and purification.
- Example 4 details the expression of human UBC in bacteria as a GST-fusion protein.
- Example 6 details the expression of a temperature-sensitive yeast UBC strain in yeast.
- the E. coli cells are weakened using lysozyme and the cells are lysed and nearly all native proteins are denatured by heating the cell suspension rapidly to 80° C. and incubating at 80°-81° C. for 20 minutes.
- the suspension is then cooled and centrifuged to precipitate the denatured proteins.
- the supernatant (containing mammalian UBC enzyme) then undergoes a high-salt polyethylene-imine treatment to precipitate nucleic acids. Centrifugation of the extract removes the nucleic acids and mammalian UBC enzyme is concentrated by use of ammonium sulfate precipitation before chromatography, preferably on a heparin-agarose column.
- the purified enzyme is at least 60% (w/w) of the protein of a preparation. Even more preferably, the protein is provided as a homogeneous preparation.
- the ubiquitin conjugating enzymes disclosed herein as having transcription repression activity may be combined with an acceptable carrier, diluent or delivery agent to form a useful composition.
- the composition has both pharmaceutical (ie, human) and non-pharmaceutical applications.
- the protein used in the composition has transcriptional repressor activity.
- the amino acid sequence of the protein includes at least a 12 amino acid portion of a ubiquitin conjugating protein such as a UBC-9 which has a transcription repression activity.
- the amino acid sequence of the protein preferably includes at least a segment of hUBC-9 or yUBC-9.
- An active-site mutant of a ubiquitin conjugating enzyme such as a cys 93 mutant of hUBC-9, whereby such a mutant lacks its ubiquitin conjugating activity, or a segment thereof, can also be used as the protein.
- a mutant in which a serine residue replaces the cysteine residue is preferred.
- the composition can further include a biochemical inhibitor suitable for inhibiting the active site cysteine of the ubiquitin conjugating enzyme.
- An exemplary suitable inhibitor in n-ethyl-maleimide An exemplary suitable inhibitor in n-ethyl-maleimide.
- the protein in the composition may have only transcriptional repressor activity, or have such an activity as well as ubiquitin conjugating activity.
- the composition may further comprise one or more other proteins, including for example a second protein having transcriptional repressor activity, such as WT1.
- the composition may also comprise other proteins having a DNA binding domain with which the ubiquitin conjugating enzyme or segment thereof interacts.
- the protein used in the composition may be a fusion protein which has a amino acid sequence that includes a DNA binding domain and a transcriptional repressor domain.
- the repressor domain of the fusion protein preferably includes at least a 12 amino acid segment of a ubiquitin conjugating enzyme having transcriptional repressor activity.
- the DNA binding domain is preferably a domain which binds to or interacts with or otherwise associates with a region of a gene which is sufficiently close to the promotor region to allow the ubiquitin conjugating enzyme or segment thereof to interact with the promoter region, and particularly, with the TATA binding protein at the TATA binding site.
- Such domains include the amino acid sequences of the Gal4 domain, the LexA domain, and the many zinc-finger domains.
- the protein is combined with a pharmaceutically acceptable carrier, diluent or gene therapy delivery agent, and a pharmaceutically active amount of the protein is used in the composition.
- the amount is preferably an amount that is effective to achieve modulation or regulation or suppression of gene transcription of a target gene. While smaller or larger amounts may be suitable in particular applications, the pharmaceutically active amount of the protein is preferably an amount sufficient to increase the concentration of the protein in the cell of the target gene being regulated by a factor ranging from about 1% to about 1000% relative to the amount of the protein which is endogenous to the cell. The increase in concentration more preferably ranges from about 10% to about 100%.
- the amount is taken relative to the endogenous amount of the ubiquitin conjugating enzyme in its natural full-sequence state.
- the particular dosage administered for a particular pharmaceutical application while preferably consistent with the aforementioned amounts, will be dependent upon the age, health, and weight of the recipient, type of concurrent treatment, if any, frequency of treatment, the nature of the effect desired, and whether a localized tissue or system-wide effect is being sought.
- a tumor-inhibiting amount is to be administered.
- an effective amount to achieve such regulation, modulation or suppression determined by the factors outlined above is to be applied.
- the amount of protein used in a non-pharmaceutical application may be in a range similar to that for pharmaceutical compositions, but may also include amounts outside this range.
- the nucleic acid polymers which encode a ubiquitin conjugating enzyme such as a UBC-9 having transcriptional repressor activity, or which encode a segment thereof, can be used in a nucleic acid composition in combination with a gene therapy delivery agent.
- gene therapy relates to operations and/or manipulations affecting both human and non-human genes, whether such operations are in-vivo or ex-vivo in nature.
- the composition preferably comprises a nucleic acid polymer that encodes a protein which has transcriptional repressor activity.
- the transcriptional repressor protein has an amino acid sequence which includes at least a portion of the amino acid sequence of a ubiquitin conjugating enzyme having transcriptional repressor activity, with the included portion being at least about 12 amino acid residues in length.
- the nucleic acid polymer can have a nucleotide sequence complementary to the nucleic acid sequence of the immediately aforementioned nucleic acid polymer.
- the ubiquitin conjugating enzyme can be a UBC-9 such as hUBC-9, or a segment thereof, or a mutant thereof lacking ubiquitin conjugating activity.
- the composition may further comprise or be used in conjunction with a biochemical inhibitor of the ubiquitin conjugating activity of the ubiquitin conjugating enzyme.
- the nucleic acid polymer can also encode a fusion protein such as the aforementioned fusion protein described in connection with the above-described protein composition.
- the nucleic acid composition comprises a pharmaceutically effective amount of the nucleic acid and a pharmaceutically acceptable gene therapy delivery means.
- the amount of nucleic acid required will vary depending on the type of cell, the effect being sought and on the delivery system used to introduce the nucleic acid polymer into a target cell.
- the amount of nucleic acid polymer is preferably an amount sufficient to, upon expression in the target cell, result in an amount of protein sufficient to regulate or modulate or repress transcription of the target gene.
- the amount is sufficient to increase the concentration of the protein in the cell of the target gene being regulated by a factor ranging from about 1% to about 1000% relative to the amount of the protein which is endogenous to the cell of the gene being regulated.
- the increase in concentration more preferably ranges from about 10% to about 100%.
- the nucleic acid polymer of the agent encodes a protein which is a segment of a ubiquitin conjugating enzyme having transcriptional repressor activity
- the amount is taken relative to the endogenous amount of the ubiquitin conjugating enzyme in its natural full-sequence state.
- Gene therapy delivery agents are used to introduce the nucleic acid polymer into target cells or to enhance the uptake of the nucleic acid polymer by the target cells.
- Several approaches for introducing the nucleic acid polymer into the cell and effecting expression thereof are known and practiced by those of skill in the art. (Mulligan, R., The Basic Science of Gene Therapy, SCIENCE, Vol. 260, pp.926-32 (1993)).
- the nucleic acid polymer of the composition may be combined, complexed, coupled or fused with a delivery agent which introduces the nucleic acid polymer into a human cell in vivo.
- the nucleic acid may be combined with a lipophilic cationic compound, which may be in the form of liposomes.
- nucleic acid may be combined with a lipophilic carrier such as any one of a number of sterols including cholesterol, cholate and deoxycholic acid.
- a lipophilic carrier such as any one of a number of sterols including cholesterol, cholate and deoxycholic acid.
- a preferred sterol is cholesterol.
- the nucleic acid may be conjugated to a peptide that is ingested by cells. Examples of useful peptides include peptide hormones or antibodies. By choosing a peptide that is selectively taken up by Wilm's tumor or other neoplastic cells, specific delivery of the nucleic acid may be effected.
- the nucleic acid may be covalently bound to the peptide via methods well known in the art.
- the peptide of choice may then be attached to the activated enzyme via an amino and sulfydryl reactive hetero bifunctional reagent. The latter is bound to a cysteine residue present in the peptide.
- the nucleic acid Upon exposure of target cells to the nucleic acid bound to the peptide, the nucleic acid is endocytosed and is rendered available for modulation of gene transcription.
- the nucleic acid polymer of the present invention can also be delivered to specific tissues using a DNA-antibody conjugate, such as is described in U.S. Pat. No. 5,428,132 to Hirsch et al.
- the gene therapy delivery agent is a construct having cDNA which includes the nucleic acid polymer and which can be expressed in a host cell.
- a construct is infected or transfected into the cell and expresses the ubiquitin conjugating enzyme having transcriptional repressor activity in the cell.
- the composition can be a virus having a viral genome which comprises the nucleic acid polymer of the agent or which is complexed to the nucleic acid polymer of the agent.
- the gene therapy delivery agent is a human cell.
- the nucleic acid polymer of the composition is inserted into a human cell in vitro and the cell comprising the nucleic acid polymer is then introduced into the body.
- the encoded ubiquitin conjugating enzyme is then expressed by the cells in vivo.
- compositions comprising the nucleic acid polymer and the pharmaceutical compositions comprising the protein of the present invention may be administered by any means that achieve their intended purpose.
- administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, or transdermal routes.
- Formulations for parenteral administration can include aqueous solutions of the composition or pharmaceutical composition in water-soluble form, for example, water-soluble salts.
- suspensions of the active compounds in oily injection suspensions may be administered.
- Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
- the suspension may also contain stabilizers.
- the particular gene therapy delivery agent used in the composition of the present invention and the determination of optimal ranges of effective amounts of each component is within capacities of a person of skill in the art of the art.
- compositions of the present invention can be used in a variety of pharmaceutical and non-pharmaceutical applications.
- gene transcription in cells can be regulated, enhanced or repressed, by controlling the concentration of UBC-9 and/or of TBP to which a target gene is exposed or in which a target gene comes in contact.
- repression of transcription can be carried out in a gene-specific manner by positioning the UBC enzymes near the promoter regions of various genes, for example, by fusion of a UBC-9 repressor domain with a gene-specific DNA binding domain, or alternatively, by protein-protein interactions between UBC-9 and proteins associated with the promoter region or involved with transcription initiation, such as WT1, TBP, or others.
- Eukaryotic cells are particularly preferred, as naturally occurring eukaryotic cells contain genes having promoter regions which include a TATA box.
- hUBC-9 or other UBC-9's or other ubiquitin conjugating enzymes having transcriptional repressor activity or segments thereof which are at least about 12 amino acid residues in length can disrupt the TATA binding protein's role in transcription initiation in such genes.
- the cells can be fungal cells (e.g. yeast cells), plant cells, non-human animal cells, non-human mammalian cells and human cells.
- Non-eukaryotic cells such as E. Coli can also be used where the cells comprise genetically engineered nucleic acid polymer constructs which include a promoter region which involves TBP for initiation of transcription.
- repressor gene products such as WT1 can be strikingly enhanced by such an approach, allowing for control of transcription of genes promoters on which WT1 is known to operate, such as IGF-II, PDGF A-chain, CSF-1 and IGF-R or others later discovered.
- the regulation of transcription can also be controlled by localized inhibition of the conjugating activity of UBC-9, for example, through a 93 cys mutant enzyme lacking such activity, or through agents which inhibit the active cite cysteine or which otherwise interrupt the proteolytic degradation pathway in a specific manner.
- the regulation of transcription is particularly useful in medical treatment, diagnostic and research applications.
- UBC-9 can be used in therapeutic compositions for inhibiting neoplastic tissue growth by itself, or in combination with known tumor suppressor proteins such as WT1. It is particularly suited to treating Wilm's tumors and to treating the other types of tumors with which WT1 suppressor gene is associated, including for example leukemia and mesothelioma. It can also be useful in controlling any number of human diseases which are causally linked to an overabundance of a certain protein. The gene from which the overabundant protein is expressed could be exposed to a UBC-9 or other ubiquitin conjugating enzymes which have repressor activity to decrease the amount of overabundant protein expressed.
- the repressor activity of a UBC-9 or of other ubiquitin conjugating enzymes could be applied to effect an increase in the expression of a particular protein of interest.
- An increase in a protein of interest can be effected through a "rebound" mechanism, where the increase therein is a result of a natural biochemical mechanism following a decrease in the amount of a second protein present in the system.
- the decrease in the amount of the second protein is accomplished according to the methods of the present invention directed to the gene which encodes that protein.
- Another significant application of the present invention includes the treatment of a human viral infection. This application would include exposing the viral genome of human virus, and particularly, the promoter region of the genome, to a ubiquitin conjugating enzyme having transcriptional repressor activity. By suppressing transcription of a viral genome, the virus may be killed or at least controlled.
- the invention could also be used to kill or at least help control yeast infections.
- transcription regulation is useful in a variety of non-human, non-pharmaceutical applications.
- the invention could, for example, be used for the treatment of animals or of particular animal diseases much as described above.
- the present invention is also useful for treating plant diseases resulting from overabundant expression, and may have other plant applications as well.
- hUBC-9 may be used to develop animal-based models or in-vitro assays.
- an animal having a selective protein deficiency can be developed by administering the pharmaceutical composition or the biochemical agent of the present invention to an animal whereby transcription of a target gene encoding the protein of interest is repressed by the repressor activity of a UBC-9 or other ubiquitin conjugating enzyme having a repressor activity.
- An alternative application could include an in-vitro comparative assay in which the effect of hUBC-9 on a culture of neoplastic cells or other cells of interest (e.g. 293 cells) is used as a standard against which the effect of other potential anti-cancer agents could be evaluated.
- Enzymatic conversion processes in which chemicals are commercially produced using enzymes expressed in cells can also take advantage of the present invention.
- Exemplary bioconversion processes include the yeast-catalyzed processes associated with the brewing and baking industries, and as well as the commercial production of a variety of carboxylic acids, including essential amino acids or analogs thereof, from amides or nitrites.
- the invention can also be used in bioconversion processes which are integral to bioremediation measures being carried out to effect environmental cleanup.
- the cells used in such enzymatic conversion processes can be eukaryotic cells or non-eukaryotic cells, such as genetically engineered E. coli cells. Other uses and applications of the several aspects of the invention will be apparent to those skilled in the art.
- the repressor domain (residues 85-179) within the N-terminal region of each of the alternative splice variants of WT1 were previously mapped and identified as functioning independently as a potent repressor when fused to a Gal4 binding domain. (Wang, Z.-Y., et al. 1993). This repressor domain was also shown to block the repressor function of WT1 if expressed independently without a functional DNA binding domain, suggesting that the repressor domain lacking DNA binding activity competed with WT1 for an interactive nuclear factor needed for WT1 to function as transcription repressor. (Wang, Z.-Y., et al., 1995).
- the interactive factor now identified as hUBC-9, was isolated by using a yeast two hybrid screen.
- a vector, LexADB-WT-N was constructed by coupling residues 85-179 of human WT1 with the Lex DNA binding domain.
- pLexADB/WT-N a cDNA fragment encoding the negative regulating domain (residues 85-179) of WT1 was obtained by digestion of the plasmid PSGWT-N with XbaI, blunt ended with Klenow fragment and the EcoRI digestion, and cloned into EcoRI and SmaI treated vector pStop116, which was modified from plasmid pBTM116 by introducing stop codons in each of three reading frames within the polylinker region.
- Yeast strain L40 was used in library screening. L40 was transformed with pLexADB/WT-N and then with the Gal4 activation domain fused with human placenta cDNA library (Clontech, Calif.) as recommended by the manufacturer. Two million yeast transformants were screened. Positive colonies on His - plates were further tested for ⁇ -Galactosidase activity with a filter assay.
- the 65 positive plasmids recovered were re-introduced to yeast to re-check specificity and for quantitation of ⁇ -gal activity.
- ⁇ -galactosidase activity units are shown, in Table 2, for the DNA-binding domain fusion partner coupled with the vector alone and with the vector fused with hUBC-9 fused with the Gal4 activation domain.
- Table 2 shows the binding specificity of hUBC-9 to the negative regulatory domain of WT1 in the yeast two hybrid system.
- LexADB-WT-N failed to activate transcription of reporter genes containing LexA binding sites in yeast when analyzed alone.
- FIG. 2A shows the Northern blots of hUBC-9 in different human tissues.
- Each lane contained 2 ⁇ g poly A + RNA from heart (H), brain (B), placenta (Pl), lung (Li), smooth muscle (SM), kidney (K), pancreas (Pa).
- the ⁇ -actin cDNA was used to probe the same blot as control. The size markers are indicated on the left side of the blot.
- FIG. 2B shows the Southern blot analysis of the hUBC-9 gene.
- hUBC-9 was fused with glutathione S-transferase (GST) by expression in bacteria as a GST-hUBC-9 fusion protein.
- GST-hUBC-9 and GST were independently coupled to a reduced glutathione sepharose matrix and washed extensively. Extracts from 293 cells which had been transfected with vectors expressing WT1 and various WT1 domains were then passed over the columns, and after incubating and washing, eluates were obtained. The eluates were separated by SDS-PAGE, transferred to nitrocellulose filter for immunoblotting, and analyzed by Western blot using anti-WT1 (1:500) and anti-IgG coupled with peroxidase. The blot was visualized by color fluorography.
- GST-hUBC-9 pBS-hUBC-9 was digested with EcoRI and the 1.1-kb insert was subcloned into the EcoR1 site of the PGEX-KG vector containing GST in frame.
- E. coli strain DH5 ⁇ was transformed with GST-hUBC-9 and GST-hUBC-9 was extracted and purified on glutathione-sepharose beads.
- GST and GST-hUBC-9 fusion protein were independently bound to glutathione-Sepharose beads and washed extensively.
- WT1 and various domains thereof were expressed in 293 cells as previously described (Wang et al., 1993). Extracts were made from 2 ⁇ 10 6 293 (human embryonic kidney cell) cells transfected with CMV promoter driven expression vectors encoding full length and the WT1 ⁇ 1-84, WT1 ⁇ 1-294, and WT1 ⁇ 297-429 domains of WT1.
- In vitro binding assays were performed by incubating the extracts with the sepharose beads containing 2-3 ⁇ g of GST and GST-hUBC-9 in lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.1% NP-40, 50 mM NaF, 1 mM PMSF, 1 ⁇ g leupeptin/ml, 1 ⁇ g antipain/ml) for 2-3 hours at room temperature.
- Complexes were washed extensively with lysis buffer and lysis buffer with 0.5M NaCl, boiled in SDS PAGE loading buffer (1% SDS, 10% ⁇ -mercaptoethanol), and run on 5% SDS-polyacylamide gels.
- FIG. 3A shows the in vitro binding of WT1 and hUBC-9.
- the left column shows the cell lysate control results with the arrow indicating WT1 at the expected estimated molecular mass of 14 kd.
- the right column shows the GST control results.
- the middle column shows the results for the GST-hUBC-9 fusion protein with the associated arrows indicating binding between WT1 and the GST-hUBC-9 matrix. Because no similar binding was observed between WT1 and the GST control matrix, these results demonstrate that WT1 binds to or associates with hUBC-9.
- An expression vector encoding the influenza virus hemagglutinin (HA) tagged hUBC-9 was constructed by cloning the 1.1 kb EcoRI fragment of hUBC-9 into the EcoRI site of expression vector PGCN (REF) in frame with a cDNA fragment encoding the HA peptide.
- 293 cells were cotransfected with WT1 and HA-tagged hUBC-9 expression plasmids.
- Cellular lysates were prepared and the extracts were immunoprecipitated with either anti-WT1 antibody or a nonspecific rabbit polyclonal antibody (anti-Gal4DB).
- WT1 associated proteins were separated on 15% SDS-PAGE and blotted. The blot was then analyzed by probing with anti-HA monoclonal antibody which recognized the HA tagged hUBC-9.
- FIG. 3B shows the results of the co-immunoprecipitation of WT1 and hUBC-9.
- Yeast strain W9432 (MATa, ubc9- ⁇ 1::TRP1, pSE362 ARS1, CEN4, HIS3!-ubc9-1) is isogenic to W303 except for carrying a replacement of the genomic yUBC-9 coding sequence by the TRP1 marker and a plasmid-borne copy of the temperature sensitive yUBC-9-1 allele (1.5 kb Xba1-Ssp1 fragment).
- hUBC-9 cDNA (1.1 kb EcoRI fragment) and yUBC-9 gene (0.6 kb EcoRI-XbaI fragment) were each fused to the Gal1 promoter in vectors p416GAL1 (ARSH4, CEN6, URA3) and pSE936 (ARS1, CEN4, URA3), respectively.
- the temperature-sensitive yeast strain (W9432) was independently transformed with the hUBC-9 (Row 1) and yUBC-9 (Row 4) control vectors (p416GAL1 and pSE936, respectively) and with a construct expressing hUBC-9 cDNA (Row 2) or the yUBC-9 gene (Row 3).
- hUBC-9 hUBC-9
- yUBC-9 yUBC-9 gene
- 293 cells were co-transfected by calcium phosphate/DNA precipitation with hUBC-9 and WT1 expression constructs under the control of the CMV promoter and with a PDGF A-chain promoter driven CAT reporter plasmid.
- FIG. 5A shows the results of the CAT assay and ⁇ -galactosidase assays. CAT activity was quantitated by scintillation counting of excised sections of TLC plates.
- FIG. 5B shows the relative CAT activity values from different assays at different times, including the standard deviation of each. The experiments demonstrate that hUBC-9 enhances the transcriptional repressor activity of WT1 in human embryonic kidney cell (293 cell).
- hUBC-9 was coupled to the Gal4 DNA binding domain and evaluated for its effect on transcription in a reporter system.
- a control expression vector pSG424, was constructed with a SV40 promoter driven Gal4 DNA binding domain.
- a fusion protein expression vector, pSG-hUBC-9 was constructed with full length cDNA of hUBC-9 fused with Gal4 DNA binding domain driven by SV40 promoter.
- pSG-hUBC-9 was constructed by inserting an EcoRl DNA fragment containing full length of hUBC-9 cDNA into the EcoRl site of expression vector pSG424.
- Reporter plasmids pSV CAT and 5 ⁇ UAS pSV CAT were provided by Dr. S. Weintraub (Washington University at St. Louis).
- the pSV CAT plasmid included a SV40 promoter fused with CAT reporter gene (Promega, Madison, Wis.).
- the 5 ⁇ UAS pSV CAT plasmid included a pSV CAT plasmid with additional 5 copies of the Gal4 binding sites upstream of the SV40 promoter.
- FIG. 6B shows the results of CAT and ⁇ -galactosidase assays, performed as described above (Example 7), for different amounts of expression plasmids, as indicated.
- CAT activity is shown as CAT activity relative to the control alone.
- hUBC-9, yUBC-9 and yUBC-9-m were independently coupled to a Gal4 DNA binding domain and evaluated for effect on transcription in a reporter system.
- control expression vector, pSG424, and hUBC-9/Gal4 fusion protein expression vector, pSG-hUBC-9 were constructed as described above (Example 8).
- a yUBC-9/Gal4 expression vector, pSG-yUBC-9 was constructed by inserting an EcoRl DNA fragment containing full length of yUBC-9 cDNA into the EcoRI site of expression vector pSG424.
- a yUBC-9-m/Gal4 expression vector, pSG-yUBC-9-m was constructed by digestion of pUC19-yUBC9-m plasmid with HindIII, blunted with Klenow fragment, and then digested by EcoRI and cloned into EcoRI-SmaI digested pSG424 plasmid.
- the reporter plasmids, depicted in FIG. 7A, were as obtained described above (Example 8).
- Example 7 The results of CAT and ⁇ -galactosidase assays, performed as discussed above (Example 7), for cotransfection experiments are shown in FIG. 7B and 7C for human and yeast UBC's, respectively. 5 ⁇ g of reporter plasmid DNA were used in each transfection with various amounts of expression plasmids, as indicated.
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Abstract
Description
TABLE 1 ______________________________________ Amino Acid Abbreviations ______________________________________ A Ala Alanine B Asx Asparagine or aspartic acid C Cys Cysteine D Asp Aspartic acid E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y Tyr Tyrosine Z Glx Glutamine or glutamic acid ______________________________________
TABLE 2 ______________________________________ Binding Specificity of hUBC-9 in Two-Hybrid System DNA-binding *-Galactosidase activity domain fusion partner Vector hUBC-9 ______________________________________ Vector alone: N.D. 0.2 ± 0.1 + WT 85-179: 0.3 ± 0.2 57.9 ± 16.2 + WT 250-266: 0.2 ± 0.2 0.3 ± 0.2 + Lamin C: 0.3 ± 0.1 1.5 ± 0.5 ______________________________________
__________________________________________________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 4 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1856 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 807..1283 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GGATGGGAAGCGAGCATGGTGAGTCCTCAAGTCGCAGCTGGGCCTGCCACGTGGGAGTGG60 AGGGTGGAGGAACGTGTGGAGTTTCGGAGTCCAGCCCAGTGCGAGACAGCCTTGAAACCG120 TGGTTGGCGGGCGCTCCACTCCGCTCTGGGCTCGAACCCTGCCTGACCCTAGCTGTGCCC180 CCCACTTTCTCCCTGTCTGGCCCCTGCTCCCCGCCCCCTCACTTAGAGGAGGGCACGGGG240 AAGGGCAAACGGTCCAGAGGGCGGGCGGCTGCGGGCTCCTCTGCATCATGTGAGGAGGGC300 GTGGGGAAGGACATCCTGGTGGGGCCCGATCTGGGCTGCCTCCAGCCCGGGCCTGTGTCT360 TGGACTTAGTCGTGGACCTGGAGGCCAGTGCCCGGCTGGCCCTGTCACCCTCTCGCTGTG420 ACGCCAGCGCCTGCTGACTGGAGGACCCAGGTTCCTTCGCCTGCTTTTTCTCAGGCTGCC480 CTGAGGATCTGTGTTTGGTGAAAAGGAGCCAAATTCACCTGCAGGGCAGGCGGCTCTAGC540 AGCTTCAGAAGCCTGGTGCCCTGGCGACACTGGACCTGCCTTGGCTTCTTTGATCCCAAC600 CCCACCCCCGATTTCTGCTCTGCTGACTGGGGAAGTCATCGTGCCACCCAGAACCTGAGT660 GCGGGCCTCTCAGAGCTCCTTCGTCCGTGGGTCTGCCGGGGACTGGGCCTTGTCTCCCTG720 GCGAGTGCCAGGTGAGGCTGCGGCGGCTCCGACGCAGGTGGAGCTGCTGACCTGGCCCCT780 TTCTGCGGCTGCGAGGGACTTTGAACATGTCGGGGATCGCCCTCAGCAGACTC833 MetSerGlyIleAlaLeuSerArgLeu 15 GCCCAGGAGAGGAAAGCATGGAGGAAAGACCACCCATTTGGTTTCGTG881 AlaGlnGluArgLysAlaTrpArgLysAspHisProPheGlyPheVal 10152025 GCTGTCCCAACAAAAAATCCCGATGGCACGATGAACCTCATGAACTGG929 AlaValProThrLysAsnProAspGlyThrMetAsnLeuMetAsnTrp 303540 GAGTGCGCCATTCCAGGAAAGAAAGGGACTCCGTGGGAAGGAGGCTTG977 GluCysAlaIleProGlyLysLysGlyThrProTrpGluGlyGlyLeu 455055 TTTAAACTACGGATGCTTTTCAAAGATGATTATCCATCTTCGCCACCA1025 PheLysLeuArgMetLeuPheLysAspAspTyrProSerSerProPro 606570 AAATGTAAATTCGAACCACCATTATTTCACCCGAATGTGTACCCTTCG1073 LysCysLysPheGluProProLeuPheHisProAsnValTyrProSer 758085 GGGACAGTGTGCCTGTCCATCTTAGAGGAGGACAAGGACTGGAGGCCA1121 GlyThrValCysLeuSerIleLeuGluGluAspLysAspTrpArgPro 9095100105 GCCATCACAATCAAACAGATCCTATTAGGAATACAGGAACTTCTAAAT1169 AlaIleThrIleLysGlnIleLeuLeuGlyIleGlnGluLeuLeuAsn 110115120 GAACCAAATATCCAAGACCCAGCTCAAGCAGAGGCCTACACGATTTAC1217 GluProAsnIleGlnAspProAlaGlnAlaGluAlaTyrThrIleTyr 125130135 TGCCAAAACAGAGTGGAGTACGAGAAAAGGGTCCGAGCACAAGCCAAG1265 CysGlnAsnArgValGluTyrGluLysArgValArgAlaGlnAlaLys 140145150 AAGTTTGCGCCCTCATAAGCAGCGACCTTGTGGCATCGTCAGAAGGAAGGGATTG1320 LysPheAlaProSer 155 GTTTGGCAAGAACTTGTTTACAACATTTTTGCAAATCTAAAGTTGCTCCATACAATGACT1380 AGTCACCTGGGGGGGTTGGGCGGGCGCCATCTTCCATTGCCGCCGCGGGTGTGCGGTCTC1440 GATTCGCTGAATTGCCCGTTTCCATACAGGGTCTCTTCCTTCGGTCTTTTGTATTTTTGA1500 TTGTTATGTAAAACTCGCTTTTATTTTAATATTGATGTCAGTATTTCAACTGCTGTAAAA1560 TTATAAACTTTTATACTTGGGTAAGTCCCCCAGGCGAGTTCCTCGCTCTGGGATGCAGGC1620 ATGCTTCTCACCGTGCAGAGCTGCACTTGGCCTCAGCTGGCTGTATGGAAATGCACCCTC1680 CCTCCTGCGCTCCTCTCTAGAACCTGGGCTGTGCTGCTTTTGAGCCTCAGACCCCAGGGC1740 AGCATCTCGGTTCTGCGCCACTTCCTTTGTGTTTATATGGCGTTTTGTCTGTGTTGCTGT1800 TTAGGTAAATAAACTGTTTATATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA1856 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1137 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 88..564 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GGGAAGCGCCGCCGCCGCCGCCCCGCTCGGTCCTCCACCTGTCCGCTACGCTCGCCGGGG60 CTGCGGCCGCCCCGAGGGACTTTGAACATGTCGGGGATCGCCCTCAGCAGA111 MetSerGlyIleAlaLeuSerArg 15 CTCGCCCAGGAGAGGAAAGCATGGAGGAAAGACCACCCATTTGGTTTC159 LeuAlaGlnGluArgLysAlaTrpArgLysAspHisProPheGlyPhe 101520 GTGGCTGTCCCAACAAAAAATCCCGATGGCACGATGAACCTCATGAAC207 ValAlaValProThrLysAsnProAspGlyThrMetAsnLeuMetAsn 25303540 TGGGAGTGCGCCATTCCAGGAAAGAAAGGGACTCCGTGGGAAGGAGGC255 TrpGluCysAlaIleProGlyLysLysGlyThrProTrpGluGlyGly 455055 TTGTTTAAACTACGGATGCTTTTCAAAGATGATTATCCATCTTCGCCA303 LeuPheLysLeuArgMetLeuPheLysAspAspTyrProSerSerPro 606570 CCAAAATGTAAATTCGAACCACCATTATTTCACCCGAATGTGTACCCT351 ProLysCysLysPheGluProProLeuPheHisProAsnValTyrPro 758085 TCGGGGACAGTGTGCCTGTCCATCTTAGAGGAGGACAAGGACTGGAGG399 SerGlyThrValCysLeuSerIleLeuGluGluAspLysAspTrpArg 9095100 CCAGCCATCACAATCAAACAGATCCTATTAGGAATACAGGAACTTCTA447 ProAlaIleThrIleLysGlnIleLeuLeuGlyIleGlnGluLeuLeu 105110115120 AATGAACCAAATATCCAAGACCCAGCTCAAGCAGAGGCCTACACGATT495 AsnGluProAsnIleGlnAspProAlaGlnAlaGluAlaTyrThrIle 125130135 TACTGCCAAAACAGAGTGGAGTACGAGAAAAGGGTCCGAGCACAAGCC543 TyrCysGlnAsnArgValGluTyrGluLysArgValArgAlaGlnAla 140145150 AAGAAGTTTGCGCCCTCATAAGCAGCGACCTTGTGGCATCGTCAGAAG591 LysLysPheAlaProSer 155 GAAGGGATTGGTTTGGCAAGAACTTGTTTACAACATTTTTGCAAATCTAAAGTTGCTCCA651 TACAATGACTAGTCACCTGGGGGGGTTGGGCGGGCGCCATCTTCCATTGCCGCCGCGGGT711 GTGCGGTCTCGATTCGCTGAATTGCCCGTTTCCATACAGGGTCTCTTCCTTCGGTCTTTT771 GTATTTTTGATTGTTATGTAAAACTCGCTTTTATTTTAATATTGATGTCAGTATTTCAAC831 TGCTGTAAAATTATAAACTTTTATACTTGGGTAAGTCCCCCAGGCGAGTTCCTCGCTCTG891 GGATGCAGGCATGCTTCTCACCGTGCAGAGCTGCACTTGGCCTCAGCTGGCTGTATGGAA951 ATGCACCCTCCCTCCTGCGCTCCTCTCTAGAACCTGGGCTGTGCTGCTTTTGAGCCTCAG1011 ACCCCAGGGCAGCATCTCGGTTCTGCGCCACTTCCTTTGTGTTTATATGGCGTTTTGTCT1071 GTGTTGCTGTTTAGGTAAATAAACTGTTTATATAAAAAAAAAAAAAAAAAAAAAAAAAAA1131 AAAAAA1137 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 158 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: MetSerGlyIleAlaLeuSerArgLeuAlaGlnGluArgLysAlaTrp 151015 ArgLysAspHisProPheGlyPheValAlaValProThrLysAsnPro 202530 AspGlyThrMetAsnLeuMetAsnTrpGluCysAlaIleProGlyLys 354045 LysGlyThrProTrpGluGlyGlyLeuPheLysLeuArgMetLeuPhe 505560 LysAspAspTyrProSerSerProProLysCysLysPheGluProPro 65707580 LeuPheHisProAsnValTyrProSerGlyThrValCysLeuSerIle 859095 LeuGluGluAspLysAspTrpArgProAlaIleThrIleLysGlnIle 100105110 LeuLeuGlyIleGlnGluLeuLeuAsnGluProAsnIleGlnAspPro 115120125 AlaGlnAlaGluAlaTyrThrIleTyrCysGlnAsnArgValGluTyr 130135140 GluLysArgValArgAlaGlnAlaLysLysPheAlaProSer 145150155 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 157 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: MetSerSerLeuCysLeuGlnArgLeuGlnGluGluArgLysLysTrp 151015 ArgLysAspHisProPheGlyPheTyrAlaLysProValLysLysAla 202530 AspGlySerMetAspLeuGlnLysTrpGluAlaGlyIleProGlyLys 354045 GluGlyThrAsnTrpAlaGlyGlyValTyrProIleThrValGluTyr 505560 ProAsnGluTyrProSerLysProProLysValLysPheProAlaGly 65707580 PheTyrHisProAsnValTyrProSerGlyThrIleCysLeuSerIle 859095 LeuAsnGluAspGlnAspTrpArgProAlaIleThrLeuLysGlnIle 100105110 ValLeuGlyValGlnAspLeuLeuAspSerProAsnProAsnSerPro 115120125 AlaGlnGluProAlaTrpArgSerPheSerArgAsnLysAlaGluTyr 130135140 AspLysLysValLeuLeuGlnAlaLysGlnTyrSerLys 145150155 __________________________________________________________________________
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US20030082561A1 (en) * | 2000-07-21 | 2003-05-01 | Takashi Sera | Zinc finger domain recognition code and uses thereof |
US20030134350A1 (en) * | 2000-07-21 | 2003-07-17 | Takashi Sera | Zinc finger domain recognition code and uses thereof |
US6808926B1 (en) | 1999-08-27 | 2004-10-26 | Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food | Repressing gene expression in plants |
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